2.1 Expression and purification of FGF2 and its variants
Recombinant human fibroblast growth factor 2 (FGF2) and its variants were expressed in the E. coli system as previously reported (Huang et al. , 2017). The Fast Mutagenesis System Kit (TransGen Biotech, Beijing, China) was used to introduce mutations into native FGF2 expression vector in order to generate the variant constructs. BL21 (DE3) competent cells were transformed with the expression vectors and cultured in LB medium with shaking at 37 °C to an absorbance of 0.5 at 600 nm, then protein expression was induced by addition of 1 mM isopropyl 1-thio-D-galactopyranoside (IPTG) and continued incubation for 4 h. Cell pellets were lysed in 150 mM NaCl, 10% glycerol, 25 mM Hepes-NaOH, pH 7.5, and 5 mM EDTA. The cell suspensions were centrifuged at 12,000 rpm for 30 min at 4 °C, and the soluble lysate fractions containing FGF2 variants proteins were purified by heparin affinity column (Hitrap Heparin HP column, 5 ml), followed by further separation by Superdex 75 10/300 gel filtration column. The purity of FGF2 variants was confirmed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified FGF2 variants were concentrated using Amicon Ultra Centrifugal Filters and the resulting concentrations were determined by absorbance measurements using a Nanodrop spectrophotometer. The detailed amino acid sequences of these proteins were shown in supplemental table 1 .